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Germinal centers (GCs) are microanatomical structures critical for the development of high-affinity Abs and B cell memory. They are organized into two zones,light and dark,with coordinated roles,controlled by local signaling. The innate lectin-like transcript 1 (LLT1) is known to be expressed on B cells,but its functional role in the GC reaction has not been explored. In this study,we report high expression of LLT1 on GC-associated B cells,early plasmablasts,and GC-derived lymphomas. LLT1 expression was readily induced via BCR,CD40,and CpG stimulation on B cells. Unexpectedly,we found high expression of the LLT1 ligand,CD161,on follicular dendritic cells. Triggering of LLT1 supported B cell activation,CD83 upregulation,and CXCR4 downregulation. Overall,these data suggest that LLT1-CD161 interactions play a novel and important role in B cell maturation within the GC in humans. View Publication

Directed differentiation of stem cells offers a scalable solution to the need for human cell models recapitulating islet biology and T2D pathogenesis. We profiled mRNA expression at 6 stages of an induced pluripotent stem cell (iPSC) model of endocrine pancreas development from 2 donors,and characterized the distinct transcriptomic profiles associated with each stage. Established regulators of endodermal lineage commitment,such as SOX17 (log2 fold change [FC] compared to iPSCs = 14.2,p-value = 4.9 × 10(-5)) and the pancreatic agenesis gene GATA6 (log2 FC = 12.1,p-value = 8.6 × 10(-5)),showed transcriptional variation consistent with their known developmental roles. However,these analyses highlighted many other genes with stage-specific expression patterns,some of which may be novel drivers or markers of islet development. For example,the leptin receptor gene,LEPR,was most highly expressed in published data from in vivo-matured cells compared to our endocrine pancreas-like cells (log2 FC = 5.5,p-value = 2.0 × 10(-12)),suggesting a role for the leptin pathway in the maturation process. Endocrine pancreas-like cells showed significant stage-selective expression of adult islet genes,including INS,ABCC8,and GLP1R,and enrichment of relevant GO-terms (e.g. insulin secretion"; odds ratio = 4.2� View Publication

Known molecular determinants of developmental plasticity are mainly transcription factors,while the extrinsic regulation of this process has been largely unexplored. Here we identify Cripto as one of the earliest epiblast markers and a key extracellular determinant of the naive and primed pluripotent states. We demonstrate that Cripto sustains mouse embryonic stem cell (ESC) self-renewal by modulating Wnt/β-catenin,whereas it maintains mouse epiblast stem cell (EpiSC) and human ESC pluripotency through Nodal/Smad2. Moreover,we provide unprecedented evidence that Cripto controls the metabolic reprogramming in ESCs to EpiSC transition. Remarkably,Cripto deficiency attenuates ESC lineage restriction in vitro and in vivo,and permits ESC transdifferentiation into trophectoderm lineage,suggesting that Cripto has earlier functions than previously recognized. All together,our studies provide novel insights into the current model of mammalian pluripotency and contribute to the understanding of the extrinsic regulation of the first cell lineage decision in the embryo. View Publication

BACKGROUND Prenatal alcohol exposure (PAE) in animal models results in excitatory-inhibitory (E/I) imbalance in neocortex due to alterations in the GABAergic interneuron (IN) differentiation and migration. Thus,E/I imbalance is a potential cause for intellectual disability in individuals with fetal alcohol spectrum disorder (FASD),but whether ethanol (EtOH) changes glutamatergic and GABAergic IN specification during human development remains unknown. Here,we created a human cellular model of PAE/FASD and tested the hypothesis that EtOH exposure during differentiation of human pluripotent stem cell-derived neurons (hPSNs) would cause the aberrant production of glutamatergic and GABAergic neurons,resulting in E/I imbalance. METHODS We applied 50 mM EtOH daily to differentiating hPSNs for 50 days to model chronic first-trimester exposure. We used quantitative polymerase chain reaction,immunocytochemical,and electrophysiological analysis to examine the effects of EtOH on hPSN specification and functional E/I balance. RESULTS We found that EtOH did not alter neural induction nor general forebrain patterning and had no effect on the expression of markers of excitatory cortical pyramidal neurons. In contrast,our data revealed highly significant changes to levels of transcripts involved with IN precursor development (e.g.,GSX2,DLX1/2/5/6,NR2F2) as well as mature IN specification (e.g.,SST,NPY). Interestingly,EtOH did not affect the number of GABAergic neurons generated nor the frequency or amplitude of miniature excitatory and inhibitory postsynaptic currents. CONCLUSIONS Similar to in vivo rodent studies,EtOH significantly and specifically altered the expression of genes involved with IN specification from hPSNs,but did not cause imbalances of synaptic excitation-inhibition. Thus,our findings corroborate previous studies pointing to aberrant neuronal differentiation as an underlying mechanism of intellectual disability in FASD. However,in contrast to rodent binge models,our chronic exposure model suggests possible compensatory mechanisms that may cause more subtle defects of network processing rather than gross alterations in total E/I balance. View Publication

A progressive increase in MECP2 protein levels is a crucial and precisely regulated event during neurodevelopment,but the underlying mechanism is unclear. We report that MECP2 is regulated post-transcriptionally during in vitro differentiation of human embryonic stem cells (hESCs) into cortical neurons. Using reporters to identify functional RNA sequences in the MECP2 3' UTR and genetic manipulations to explore the role of interacting factors on endogenous MECP2,we discover combinatorial mechanisms that regulate RNA stability and translation. The RNA-binding protein PUM1 and pluripotent-specific microRNAs destabilize the long MECP2 3' UTR in hESCs. Hence,the 3' UTR appears to lengthen during differentiation as the long isoform becomes stable in neurons. Meanwhile,translation of MECP2 is repressed by TIA1 in hESCs until HuC predominates in neurons,resulting in a switch to translational enhancement. Ultimately,3' UTR-directed translational fine-tuning differentially modulates MECP2 protein in the two cell types to levels appropriate for normal neurodevelopment. View Publication

Wnt/β-catenin signalling has a variety of roles in regulating stem cell fates. Its specific role in mouse epiblast stem cell self-renewal,however,remains poorly understood. Here we show that Wnt/β-catenin functions in both self-renewal and differentiation in mouse epiblast stem cells. Stabilization and nuclear translocation of β-catenin and its subsequent binding to T-cell factors induces differentiation. Conversely,retention of stabilized β-catenin in the cytoplasm maintains self-renewal. Cytoplasmic retention of β-catenin is effected by stabilization of Axin2,a downstream target of β-catenin,or by genetic modifications to β-catenin that prevent its nuclear translocation. We also find that human embryonic stem cell and mouse epiblast stem cell fates are regulated by β-catenin through similar mechanisms. Our results elucidate a new role for β-catenin in stem cell self-renewal that is independent of its transcriptional activity and will have broad implications in understanding the molecular regulation of stem cell fate. View Publication

Human embryonic stem (hES) cells hold great promise for application of human cell and tissue replacement therapy. However,the overwhelming majority of currently available hES cell lines have been directly or indirectly exposed to materials containing animal-derived components during their derivation,propagation,and cryopreservation. Unlike feeder based cultures,which require the simultaneous growth of feeder and stem cells,resulting in mixed cell populations,stem cells grown on feeder-free systems are easily separated from the surface,presenting a pure population of cells for downstream applications. In this study we have developed a novel method to expand hES cells in xeno-free,feeder-free conditions using two different matrices derived from xeno-free human foreskin fibroblasts (XF-HFFs). Using XF-HFF-derived extracellular matrix,together with 100ng/ml recombinant bFGF supplemented HEScGRO Basal Medium,long term xeno-free expansion of hES cells is possible. Resulting hES cells were subjected to stringent tests and were found to maintain ES cell features,including morphology,pluripotency,stable karyotype,and expression of cell surface markers,for at least 20 passages. Xeno-free culturing practices are essential for the translation of basic hES cell research into the clinic. Therefore,the method presented in this study demonstrates that hES cells can be cultured in complete xeno-free conditions without the loss of pluripotency and furthermore,without the possibility of contamination from exogenous sources. View Publication

Defining the molecular mechanisms underpinning fetal (gamma) globin gene silencing may provide strategies for reactivation of gamma-gene expression,a major therapeutic objective in patients with beta-thalassemia and sickle cell disease (SCD). We have previously demonstrated that symmetric methylation of histone H4 Arginine 3 (H4R3me2s) by the protein arginine methyltransferase PRMT5 is required for recruitment of the DNA methyltransferase DNMT3A to the gamma-promoter,and subsequent DNA methylation and gene silencing. Here we show in an erythroid cell line,and in primary adult erythroid progenitors that PRMT5 induces additional repressive epigenetic marks at the gamma-promoter through the assembly of a multiprotein repressor complex containing the histone modifying enzymes SUV4-20h1,casein kinase 2alpha (CK2alpha),and components of the nucleosome remodeling and histone deacetylation complex. Expression of a mutant form of PRMT5 lacking methyltransferase activity or shRNA-mediated knockdown of SUV4-20h1 resulted in loss of complex binding to the gamma-promoter,reversal of both histone and DNA repressive epigenetic marks,and increased gamma-gene expression. The repressive H4K20me3 mark induced by SUV4-20h1 is enriched on the gamma-promoter in erythroid progenitors from adult bone marrow compared with cord blood,suggesting developmental specificity. These studies define coordinated epigenetic events linked to fetal globin gene silencing,and provide potential therapeutic targets for the treatment of beta-thalassemia and SCD. View Publication

The discovery of induced pluripotent stem cells (iPSCs) and their application to patient-specific disease models offers new opportunities for studying the pathophysiology of neurological disorders. However,current methods for culturing iPSC-derived neuronal cells result in clustering of neurons,which precludes the analysis of individual neurons and defined neuronal networks. To address this challenge,cultures of human neurons on micropatterned surfaces are developed that promote neuronal survival over extended periods of time. This approach facilitates studies of neuronal development,cellular trafficking,and related mechanisms that require assessment of individual neurons and specific network connections. Importantly,micropatterns support the long-term stability of cultured neurons,which enables time-dependent analysis of cellular processes in living neurons. The approach described in this paper allows mechanistic studies of human neurons,both in terms of normal neuronal development and function,as well as time-dependent pathological processes,and provides a platform for testing of new therapeutics in neuropsychiatric disorders. View Publication

BACKGROUND The inhibition of Janus kinases (JAKs) and subsequent signal transducers and activators of transcription (STATs) by tofacitinib represents a new therapeutic strategy in inflammatory bowel diseases (IBD) as clinical trials have led to approval of tofacitinib for ulcerative colitis (UC) and hint at a possible efficacy for Crohn`s disease (CD). However,the impact of tofacitinib on cellular response of monocytes,which are key players in inflammatory responses,has not been investigated so far. We aimed to analyze JAK/STAT-inhibition by tofacitinib in monocytes of IBD patients and healthy controls. METHODS Primary monocytes of IBD patients with active disease and healthy controls (n = 18) were analyzed for cytokine expression and phenotype after granulocyte macrophage colony-stimulating factor (GM-CSF)/interferon (IFN)$\gamma$-stimulation and tofacitinib pretreatment (1-1000 nM) and capacity to induce Foxp3+-regulatory T cells (Tregs) in cocultures. In total,20 UC patients and 21 CD patients were included. Additionally,dose-dependent inhibition of JAK/STAT-phosphorylation was analyzed in controls. RESULTS Pro-inflammatory costimulation with GM-CSF/IFN$\gamma$ resulted in significant tumor necrosis factor (TNF$\alpha$) and interleukin (IL)-6 increase,whereas IL-10 expression decreased in monocytes. Tofacitinib modulated the responses of activated monocytes toward a regulatory phenotype through reduced TNF$\alpha$ and IL-6 secretion and enhanced Treg induction in cocultures. However,in monocytes from active IBD patients,higher tofacitinib dosages were needed for blockade of pro-inflammatory cytokines. Tofacitinib induced stronger regulatory phenotypes in monocytes of UC patients,including more effective inhibition of pro-inflammatory pathways and better restoration of anti-inflammatory mechanisms as compared with CD-derived monocytes. CONCLUSION Tofacitinib dose-dependently reprograms monocytes toward a more regulatory cell type. This beneficial effect possibly results from selective JAK/STAT-blockade by adequate tofacitinib dosage with inhibition of pro-inflammatory responses and permission of a balance-shift toward regulatory pathways. View Publication

Neurogenin3 (NEUROG3) is required for endocrine lineage formation of the pancreas and intestine. Patients with NEUROG3 mutations are born with congenital malabsorptive diarrhea due to complete loss of enteroendocrine cells,whereas endocrine pancreas development varies in an allele-specific manner. These findings suggest a context-dependent requirement for NEUROG3 in pancreas versus intestine. We utilized human tissue differentiated from NEUROG3-/- pluripotent stem cells for functional analyses. Most disease-associated alleles had hypomorphic or null phenotype in both tissues,whereas the S171fsX68 mutation had reduced activity in the pancreas but largely null in the intestine. Biochemical studies revealed NEUROG3 variants have distinct molecular defects with altered protein stability,DNA binding,and gene transcription. Moreover,NEUROG3 was highly unstable in the intestinal epithelium,explaining the enhanced sensitivity of intestinal defects relative to the pancreas. These studies emphasize that studies of human mutations in the endogenous tissue context may be required to assess structure-function relationships. View Publication

Viral vector delivery of gene therapy represents a promising approach for the treatment of numerous retinal diseases. Adeno-associated viral vectors (AAV) constitute the primary gene delivery platform; however,their limited cargo capacity restricts the delivery of several clinically relevant retinal genes. In this study,we explore the feasibility of employing high-capacity adenoviral vectors (HC-AdVs) as alternative delivery vehicles,which,with a capacity of up to 36 kb,can potentially accommodate all known retinal gene coding sequences. We utilized HC-AdVs based on the classical adenoviral type 5 (AdV5) and on a fiber-modified AdV5.F50 version,both engineered to deliver a 29.6 kb vector genome encoding a fluorescent reporter construct. The tropism of these HC-AdVs was evaluated in an induced pluripotent stem cell (iPSC)-derived human retinal organoid model. Both vector types demonstrated robust transduction efficiency,with sustained transgene expression observed for up to 110 days post-transduction. Moreover,we found efficient transduction of photoreceptors and Müller glial cells,without evidence of reactive gliosis or loss of photoreceptor cell nuclei. However,an increase in the thickness of the photoreceptor outer nuclear layer was observed at 110 days post-transduction,suggesting potential unfavorable effects on Müller glial or photoreceptor cells associated with HC-AdV transduction and/or long-term reporter overexpression. These findings suggest that while HC-AdVs show promise for large retinal gene delivery,further investigations are required to assess their long-term safety and efficacy. View Publication